Most artificial substrates for hydrolytic enzymes in current large-scale applications in diagnostic microbiology are either chromogenic or fluorogenic. Fluorogenic assays suffer from certain disadvantages, including intrinsic background fluorescence from certain samples. However, perhaps their main disadvantage is the need to use a lamp or other source of UV light to generate the fluorescence. Chromogenic enzyme substrates have an advantage in that the endpoint can be determined with the naked eye. Alternatively, the released chromogen may be assayed using simple spectrophotometers working by absorption of light in the visible wavelengths. For an enzyme substrate to be of real value in diagnostic microbiology, certain conditions need to be met. When attached to the target residue (i.e. a sugar, ester or phosphate) the artificial enzyme substrates should be practically colourless or have a very low background colouration that does not interfere significantly with the test procedure. However, once cleaved from the target portion by enzymatic hydrolysis, the free core molecule is either highly coloured or can be converted to a coloured compound in situ by further chemical (i.e., non-enzymatic) reaction. Ideally, this reaction should be virtually instantaneous with the enzymatic cleavage and the conditions or reagents required to produce the colour should preferably be already present in the media, and therefore must be able to allow adequate microbial growth. Under these conditions, the presence of the coloured end-product gives a good indication of the enzyme activity targeted. The substrates should be convenient to synthesise from inexpensive starting materials so that many different substrates can be produced from the same core molecule; they should be easy to use, suitable for continuous assays, and they should be able to work under both aerobic and anaerobic conditions. Although not a prerequisite for ultimate utility, it would be a further advantage if the chromogen was contrasting in colour to the chromogens of currently available enzyme substrates. In liquid media the chromogen should be largely soluble. In solid or gelled plate media (such as the commonly used agar plates) the chromogen should be non-diffusible so that the colour remains concentrated in the colony mass. In agar tube media, diffusion of the chromogen is acceptable.
Many different artificial chromogenic enzyme substrates derived from various core molecules have been produced and are currently commercially available. However, all core molecules have limitations as well as advantages depending upon the specific application. Some of the positive and negative attributes of the main types of chromogenic enzyme substrates employed to detect glycosidase activities may be set out as follows.
Nitrophenyl substrates are widely employed in liquid media. One common example, o-nitrophenyl-β-D-galactopyranoside (ONPG), is cheap and easy to use. However, in agar plate media diffusion of the yellow o-nitrophenol chromogen makes it impractical to detect enzyme-positive from enzyme-negative cultures of microorganisms in a polymicrobial culture. A further disadvantage is that the pale yellow colour given after hydrolysis is not dissimilar to the background colour already present in certain culture media. Moreover, the maximum colour of o-nitrophenol is only generated at highly alkaline pH at which most microorganisms will not grow. p-Nitrophenyl-β-D-glucuronide is an enzyme substrate that has been used to detect β-D-glucuronidase from E. coli and thereby identify this bacterium, but the yellow p-nitrophenol shares all the defects of its isomer o-nitrophenol. Phenolphthalein is another inexpensive core molecule of some chromogenic enzyme substrates. As with the nitrophenols, the phenolphthalein aglycone diffuses greatly in agar media. Moreover, the free phenolphthalein has to be made highly basic before the red colour develops, so phenolphthalein substrates are unsuitable for continuous assays. Although this core molecule is inexpensive, its glycosides, such as phenolphthalein-β-D-glucuronide, are very expensive, undoubtedly because of difficulties with their synthesis. For all the above reasons, phenol phthalein-derived enzyme substrates are little used currently. Resorufin is a very costly core molecule that is both chromogenic and fluorogenic; the few commercially available glycosides derived from it are thus very expensive. Resorufin substrates are suitable for liquid media only.
For use in solid media (agar plates) and other situations in which an essentially insoluble or non-diffusible chromogenic endpoint is required, indoxyl substrates tend to be preferred, X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) is a common example. On enzymatic cleavage, oxidation of 5-bromo-4-chloroindoxyl in situ yields a bright blue-green insoluble indigo dye that will stay localised within microbial colonies, thereby affording good differentiation of colonies within a polymicrobial culture on agar plates. X-β-D-glucuronide is extensively used in microbiological media to detect E. coli. A whole range of differently substituted indoxyl glycosides, carboxylic acid esters and phosphates is commercially available. The use of different coloured indoxyl substrates in the same medium has been exploited for the simultaneous detection of two or more different enzyme activities [J. N. Roth and W. J. Ferguson, U.S. Pat. No. 5,219,922, (1993); W. J. Ferguson, U.S. Pat. No. 5,358,854, (1994)]. Negative aspects of indoxyl substrates are that they are not well suited to liquid media and that the indigo dye can only be generated under oxidative conditions. This last property makes them unsuited to the detection of anaerobes and is a severe limitation when dealing with this type of organism. Indoxyl substrates have also been shown to exhibit toxicity to bacteria as evidenced by the formation of small colonies [A. S. Baron and F. E. Nano, Mol. Microbiol., 29, 247-259, (1998); L. Butterworth et al, J. Appl. Microbiol., 96, 170-176, (2004)]. Typically, indoxyl substrates are more expensive than their nitrophenyl counterparts; X-Gal is approximately one order of magnitude more expensive than ONPG in bulk.
Enzyme substrates derived from 1-naphthol, 2-naphthol, and compounds in the naphthol-AS series are known. The released naphthols are not themselves chromogenic, and a diazonium salt has to be added to produce the insoluble dye. The need to add a coupling reagent post-incubation renders these substrates unsuitable for continuous assay as well as greatly adding to the inconvenience of any test. Above all, diazonium salts are to be avoided for routine work because of their general toxicity.
James and co-workers [A. L. James et al, Applied and Environmental Microbiology, 66, 5521-5523, (2000)] synthesised the novel chromogenic substrate p-naphtholbenzein-β-D-galactopyranoside for use in solid plate media. Bacterial hydrolysis of this substrate gave pink, non-diffusible colonies. The principle on which this aglycone works appears to be its large size combined with its hydrophobic nature. Its sensitivity when challenged with nearly 400 bacterial strains was lower than X-Gal. This may explain why the substrate is not commercially available and why other glycosides of p-naphtholbenzein have not so far been reported.
Recently, substrates generating their colour via intermolecular or intramolecular aldol reactions have been disclosed [U. Spitz et al, EP 20090159639, (2009)]. Although a number of advantages are claimed for these Aldol substrates, their synthesis actually starts from other indoxyls substrates, thus making them potentially very expensive. Another disadvantage of their synthesis is that some examples require the preparation and use of some reagents not commercially available.
Artificial chromogenic enzyme substrates based on metal chelation are well-known. Glycosides of 8-hydroxyquinoline generate insoluble coloured iron chelates after release of the aglycone. A commercial medium using 8-hydroxyquinoline-β-D-glucuronide has been evaluated [R. D. Reinders et al, Lett. Appl. Microbiol., 30, 411-414, (2000)], but for bacterial diagnosis a restricting factor is the toxicity of the aglycone to Gram-positive organisms [J. D. Perry et al, J. Appl. Microbiol., 102, 410-415, (2007)]. The range of 8-hydroxyquinoline substrates commercially available is limited, possibly on account of difficulties in their synthesis, and the substrates are unsuited to liquid media. However, most of the artificial enzyme substrates described as working via metal chelation are composed of core molecules that contain an ortho-dihydroxyaromatic system. Many different compounds containing the ortho-dihydroxyaromatic system have been described as metal chelators, and they form coloured complexes with a wide variety of metal ions depending on the compound and the metal ion in question. Nevertheless, the first chromogenic chelating-type enzyme substrate containing an ortho-dihydroxyaromatic moiety to be used was a natural compound, esculin. Esculin is the β-D-glucopyranoside of esculetin. Used with an iron salt, esculin finds employment as a reagent for Group D streptococci [A. Swan, J. Clin. Path., 7, 160-163, (1954)]. Unfortunately, extensive diffusion of the esculetin-iron chromogen presents a problem on agar plate media. The core molecule esculetin is expensive, and this has undoubtedly blocked the commercial development of further glycosides made from it. Therefore esculetin glycosides are not ideal substrates.
In order to address some of the aforementioned limitations of both the indoxyl enzyme substrates and the existing substrates working by metal chelation, James and Armstrong devised novel chromogenic enzyme substrates, the lead core molecule being cyclohexenoesculetin (CHE) [A. L. James and L. Armstrong, U.S. Pat. No. 6,008,008, (1999)]. CHE contains the ortho-dihydroxyaromatic moiety. CHE substrates present no diffusion, and employing them to detect bacteria on solid plate media produces discrete black colonies in the presence of iron, which greatly assists in identification in mixed cultures. CHE enzyme substrates have no background coloration, do not show any measurable toxicity to microorganisms and they can be used under both anaerobic and aerobic conditions. The intense black colour of the CHE-iron chelate may even be used to good effect to mask the colour generated by indoxyl substrates, depending on the circumstances [J. D. Perry et al, J. Clin. Microbiol., 37, 766-768, (1999)]. One disadvantage of CHE glycosides is the relative expense of synthesising CHE itself. Metal chelating-type enzyme substrates with the ortho-dihydroxyaromatic system have also been made from the well-known dye alizarin [L. Armstrong and A. James, U.S. Pat. No. 7,052,863 (2006) and U.S. Pat. No. 7,563,592 (2009)]. Alizarin-β-D-galactopyranoside was shown to be a highly sensitive substrate in agar plate media with an optimal concentration just over half that of X-Gal in the application studied, and this concentration was very much less than that required for the galactosides of CHE and 8-hydroxyquinoline [A. L. James et al, Letters Appl. Microbiol., 30, 336, (2000)]. Another useful attribute of this substrate is its ability to form different colours of chelate depending on the metal ion used. In agar media, alizarin substrates give a violet colour with iron and a bright pink chelate with aluminium. However, the toxicity of alizarin gives rise to small colonies [J. D. Perry et al, J. Appl. Microbiol., 102, 410 (2006)].
Yet a further class of enzyme substrates employing metal chelation with an ortho-dihydroxyaromatic system has been disclosed [M. Burton, EP 1438423, (2007)]. The essence of these substrates is that the ortho-dihydroxybenzene ring is not fused to any other ring system, therefore they are catechols. Other groups (if any) are attached to the catechol ring by single bonds. Substrates of the parent compound, catechol itself, generate a fairly intense black chelate in the presence of iron salts after enzymatic hydrolysis, although this chelate is prone to diffuse in agar media. Catechol β-D-ribofuranoside was demonstrated as an effective enzyme substrate for the revelation of R-D-ribofuranosidase activity in Shigella and Salmonella, but diffusion of the chromogen would appear to limit its use on agar plate media. The catechol-derivative 3′,4′-dihydroxyflavone (DHF) affords substrates showing little or no diffusion in agar media. Like CHE, DHF substrates yield dark brown or black iron chelates with iron salts, but they show some advantage over CHE substrates in that they can form a yellow, non-diffusible chelate with aluminium salts. DHF substrates are essentially non-toxic to microorganisms, but the DHF aglycone is difficult to synthesise and, although it is commercially available, it is very expensive.
WO2008/004788 discloses the synthesis of the dicaprylate of 2,3-dihydroxynaphthalene (DHN). The compounds are said to have potential therapeutic utility for treating the skin disease caused by excessive production of melanin.
Bogdanov et al in Phosphorous, Sulphur and Silicon (2008) 183:650-651 describe synthesis of monophosphate esters of DHN and a ring brominated analogue. Uses of the esters are not disclosed.
GB2022267 discloses DHN conjugates for use in photography in conjunction with chromogenic compounds. The DHN conjugates may decompose under the action of thermal energy, or by reaction with gaseous or liquid chemicals, to then react with the chromogenic compounds to form a coloured image.
At the present time, there is no class of chromogenic enzyme substrates that is inexpensive and simple to prepare, is easy to use, and is suitable for continuous assays of microorganisms in both liquid and solid media, and under both aerobic and anaerobic conditions.